Method and composition for selectively inhibiting melanoma

ABSTRACT

A composition and method of preventing or inhibiting tumor growth, and of treating malignant melanoma, without toxic side effects are disclosed. Betulinic acid or a betulinic acid derivative is the active compound of the composition, which is topically applied to the situs of tumor.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part application of U.S. patentapplication Ser. No. 08/407,756, filed on Mar. 21, 1995, now U.S. Pat.No. ______.

[0002] This invention was made with government support under U01 CA52956awarded by the National Cancer Institute. The government has certainrights in the invention.

FIELD OF THE INVENTION

[0003] This invention relates to compositions and methods of selectivelyinhibiting tumors and, more particularly, to treating a malignantmelanoma using plant-derived compounds and derivatives thereof.

BACKGROUND OF THE INVENTION

[0004] Over the past four decades the incidence of melanoma has beenincreasing at a higher rate than any other type of cancer. It is nowtheorized that one in 90 American Caucasians will develop malignantmelanoma in their lifetime. While an increasing proportion of melanomasare diagnosed sufficiently early to respond to surgical treatment andachieve a greater than 90% ten-year survival rate, it is estimated thatnearly 7,000 individuals suffering from metastatic melanoma will die inthe United States this year.

[0005] For patients with metastatic melanoma not amenable to surgicalextirpation, treatment options are limited.5-(3,3-Dimethyl-1-triazenyl)-1-H-imidazole-4-carboxamide (dacarbazine,DTIC) is the most efficacious single chemotherapeutic agent for melanomahaving an overall response rate of 24%. But the duration of response toDTIC is generally quite poor. Combination therapy with other syntheticand recombinant agents, including N,N′-bis(2-chloroethyl)-N-nitrosurea(carmustine, BCNU), cisplatin, tamoxifen, interferon-alpha (INF-α) andinterleukin-2 (IL-2), has a higher response rate (e.g., 30-50%) in sometrials, but a durable complete response rate is uncommon and toxicity isincreased. Sequential chemotherapy has promise, but, clearly, currenttreatment options for individuals suffering from metastatic melanoma areunsatisfactory.

[0006] Various drugs derived from natural products, such as adriamycin(doxorubicin) derivatives, bleomycin, etoposide, and vincristine, andtheir derivatives, have been tested for efficacy against melanoma eitheras single agents or in combination therapy. However, similar to thesynthetic and recombinant compounds, these compounds exhibit lowresponse rates, transient complete responses, and high toxicities.

[0007] Nonetheless, as demonstrated by known and presently-used cancerchemotherapeutic agents, plant-derived natural products are a provensource of effective drugs. Two such useful natural product drugs arepaclitaxel (taxol) and camptothecin. Paclitaxel originally derived fromthe bark of the Pacific yew tree Taxus brevifolia Nutt. (Taxaceae),currently is used for the treatment of refractory or residual ovariancancer. More recently, clinical trials have been performed toinvestigate the possible role of paclitaxel in the treatment ofmetastatic melanoma. As a single agent, taxol displays activitycomparable to cisplatin and IL-2. Taxol functions by a unique mode ofaction, and promotes the polymerization of tubulin. Thus, the antitumorresponse mediated by taxol is due to its antimitotic activity. Thesecond drug of prominence, camptothecin, was isolated from the stem barkof a Chinese tree, Camptotheca acuminata Decaisne (Nyssaceae).Camptothecin also functions by a novel mechanism of action, i.e., theinhibition of topoisomerase I. Phase II trials of a water-solublecamptothecin pro-drug analog, Irinotican (CPT-11), have been completedin Japan against a variety of tumors with response rates ranging from 0%(lymphoma) to 50% (small cell lung). Topotecan, another water-solublecamptothecin analog, currently is undergoing Phase II clinical trials inthe United States.

[0008] Previous antitumor data from various animal models utilizingbetulinic acid have been extremely variable and apparently inconsistent.For example, betulinic acid was reported to demonstrate dose-dependentactivity against the Walker 256 murine carcinosarcoma tumor system atdose levels of 300 and 500 mg/kg (milligrams per kilogram) body weight.In contrast, a subsequent report indicated the compound was inactive inthe Walker 256 (400 mg/kg) and in the L1210 murine lymphocytic leukemia(200 mg/kg) models. Tests conducted at the National Cancer Instituteconfirmed these negative data.

[0009] Similarly, antitumor activity of betulinic acid in the P-388murine lymphocyte test system has been suggested. However, activity wasnot supported by tests conducted by the National Cancer Institute. Morerecently, betulinic acid was shown to block phorbol ester-inducedinflammation and epidermal ornithine decarboxylase accumulation in themouse ear model. Consistent with these observations, the carcinogenicresponse in the two-stage mouse skin model was inhibited. Thus, someweak indications of antitumor activity by betulinic acid have beenreported, but, until the present invention, no previous reports or datasuggested that betulinic acid was useful for the selective control ortreatment of human melanoma. Furthermore, to date, no information hasbeen published with respect to the selective activity of derivatives ofbetulinic acid against melanoma cells.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method and composition forpreventing or inhibiting tumor growth. The active compound is betulinicacid or a derivative of betulinic acid. The betulinic acid is isolatedby a method comprising the steps of preparing an extract from the stembark of Ziziphus mauritiana and isolating the betulinic acid.Alternatively, betulin can be isolated from the extract and used asprecursor for betulinic acid, which is prepared from betulin by a seriesof synthetic steps. The betulinic acid can be isolated from the extractby mediating a selective cytotoxic profile against human melanoma in asubject panel of human cancer cell lines, conducting a bioassay-directedfractionation based on the profile of biological activity using culturedhuman melanoma cells (MEL-2) as the monitor, and obtaining betulinicacid therefrom as the active compound. The resulting betulinic acid canbe used to prevent or inhibit tumor growth, or can be converted to aderivative to prevent or inhibit tumor growth.

[0011] An important aspect of the present invention, therefore, is toprovide a method and composition for preventing or inhibiting tumorgrowth and, particularly, for preventing or inhibiting the growth ofmelanoma using a natural product-derived compound, or a derivativethereof.

[0012] Another aspect of the present invention is to provide a treatmentmethod using betulinic acid to prevent the growth or spread of cancerouscells, wherein the betulinic acid, or a derivative thereof, is appliedin a topical preparation.

[0013] Another aspect of the present invention is to overcome theproblem of high mammalian toxicity associated with synthetic anticanceragents by using a natural product-derived compound, e.g., betulinic acidor a derivative thereof.

[0014] Still another aspect of the present invention is to overcome theproblem of insufficient availability associated with syntheticanticancer agents by utilizing readily available, and naturallyoccurring betulinic acid, or a derivative thereof.

[0015] Yet another aspect of the present invention is to preparederivatives of betulinic acid that have a highly selective activityagainst melanoma cells, and that have physical properties that make thederivatives easier to incorporate into topical preparations useful forthe prevention or inhibition of melanoma cell growth.

[0016] These and other aspects of the present invention will becomeapparent from the following description of the invention, which areintended to limit neither the spirit or scope of the invention but areonly offered as illustrations of the preferred embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a plot of mean tumor volume (in cubic centimeters (cm³))vs. time for nonestablished MEL-2 tumors in control mice and micetreated with increasing dosages of betulinic acid;

[0018]FIG. 2 is a plot of mean tumor volume (in cm³) vs. time forestablished MEL-2 tumors in control mice and mice treated with DTIC orbetulinic acid;

[0019]FIG. 3(A) is a plot of the 50 Kbp (kilobase pairs) band as % totalDNA v. time for treatment of MEL-2 cells with 2 μg/ml (micrograms permilliliter) betulinic acid;

[0020]FIG. 3(B) is a plot of the 50 Kbp band as % total DNA versusconcentration of betulinic acid (μg/ml); and

[0021]FIGS. 4 and 5 are plots of mean tumor volume (cm³) vs. time forestablished and nonestablished MEL-1 tumors in control mice and micetreated with increasing doses of betulinic acid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Betulinic acid, 3β-hydroxy-lup-20(29)-ene-28-oic acid, is anatural product isolated from several genus of higher plants. Through abioassay-directed fractionation of the stem bark of Ziziphus mauritianaLam. (Rhamnaceae), betulinic acid, a pentacyclic triterpene, wasisolated as an active compound that showed a selective cytotoxicityagainst cultured human melanoma cells. The cell lines evaluated forcytotoxicity were A431 (squamous), BC-1 (breast), COL-2 (colon), HT-1080(sarcoma), KB (human oral epidermoid carcinoma), LNCaP (prostate), LU-1(lung), U373 (glioma), and MEL-1, -2, -3, and -4 (melanoma). Betulinicacid was found to be an excellent antitumor compound against humanmelanoma due to its unique in vitro and in vivo cytotoxicity profile.Betulinic acid has shown a strong selective antitumor activity againstmelanoma by induction of apoptosis. The selective cytotoxicity ofbetulinic acid, and its lack of toxicity toward normal cells, afford afavorable therapeutic index. In addition, betulinic acid has beenreported to have an anti-HIV activity.

[0023] The bark of white birch, Betula alba, contains betulin (up toabout 25&), lup-20(29)-ene-3β,28-diol, and betulinic acid (0.025%), butit is difficult to isolate a sufficient quantity of betulinic acid toperform an extensive bioassay. It has been found that a quantity ofbetulinic acid could be provided from betulin through a simple syntheticapproach. A number of multi-step synthetic conversions of betulin tobetulinic acid have been reported, but these synthetic sequences sufferfrom a low overall yield. A concise two-step conversion of betulin tobetulinic acid, in good yield, has been reported in SyntheticCommunications, 27(9), pp. 1607-1612 (1997).

[0024] As shown in Table 1, in vitro growth of MEL-2 cells was inhibitedby betulinic acid, i.e., an ED₅₀ value of about 2 μg/ml. However, noneof the other cancer cell lines tested was affected by betulinic acid(i.e., ED₅₀ values of greater than 20 μg/ml). Such clearly definedcell-type specificity demonstrated by betulinic acid is both new andunexpected.

[0025] For example, as illustrated in Table 1, other known antitumoragents, such as paclitaxel, camptothecin, ellipticine,homoharringtonine, mithramycin A, podopyllotoxin, vinblastine andvincristine, demonstrated relatively intense, nonselective cytotoxicactivity with no discernible cell-type selectivity. Moreover, thecytotoxic response mediated by betulinic acid is not exclusively limitedto the MEL-2 melanoma cell line. Dose-response studies performed withadditional human melanoma cell lines, designated MEL-1, MEL-3 and MEL-4,demonstrated ED₅₀ values of 1.1, 3.3 and 4.8 μg/ml, respectively.

[0026] In the following Table 1, the extracted betulinic acid and theother pure compounds were tested for cycotoxity against the followingcultured human cell lines: A431 (squamous cells), BC-1 (breast), COL-2(colon), HT-1080 (sarcoma), KB (human oral epidermoid carcinoma), LNCaP(prostate), LU-1 (lung), MEL-2 (melanoma), U373 (glioma) and ZR-75-1(breast). TABLE 1 Cytotoxic Activity Profile of the Crude Ethyl AcetateExtract Obtained from Ziziphus mauritiana, Betulinic acid, OtherAntineoplastic Agents ED₅₀ (μg/ml) Compound A431 BC-1 COL-2 HT-1080 KBLNCaP LU-1 MEL-2 U373 ZR 75-1 Ziziphus mauritiana >20 >20 >209.5 >20 >20 5.2 3.7 >20 15.8 crude extract Betulinicacid >20 >20 >20 >20 >20 >20 >20 2.0 >20 >20 Taxol 0.00 0.02 0.02 0.000.02 0.02 0.00 0.06 0.008 0.02 Camptothecin 0.00 0.07 0.005 0.01 0.000.006 0.00 0.02 0.000 0.001 Ellipticine 0.5 0.2 0.3 1.8 0.04 0.8 0.020.9 1.6 0.9 Homoharringtonine 0.02 0.03 0.1 0.01 0.00 0.03 0.03 0.04 0.20.06 Mithramycin A 0.09 0.3 0.06 1.5 0.09 0.05 0.2 1.2 0.04 0.2Podophyllotoxin 0.03 0.03 0.005 0.00 0.08 0.04 0.00 0.003 0.004 0.4Vinbiastine 0.05 0.06 0.01 0.02 0.04 0.1 0.02 0.01 1.1 0.3 Vincristine0.01 0.01 0.02 0.02 0.00 0.1 0.05 0.02 0.06 0.4

[0027] Betulinic acid (1) has the structural formula:

[0028] Betulinic acid is fairly widespread in the plant kingdom, and, asa compound frequently encountered, some previous biological activitieshave been reported.

[0029] Betulinic acid was obtained by extracting a sample of air-dried,milled stem bark (450 g) of Z. mauritiana with 80% aqueous methanol. Theaqueous methanol extract then was partitioned successively with hexaneand ethyl acetate to provide hexane, ethyl acetate and aqueous extracts.Among these extracts, the ethyl acetate (13.5 g) extract showedcytotoxic activity against a cultured melanoma cell line (MEL-2) with anED₅₀ of 3.7 μg/ml. The ethyl acetate extract was chromatographed on asilica gel column using hexane-ethyl acetate (4:1 to 1:4) as eluent togive 10 fractions. Fractions 3 and 4 were combined and subjected tofurther fractionation to afford an active fraction (fraction 16) showinga major single spot by thin-layer chromatography [R_(f) 0.67: CHCl₃:MeOH(chloroform:methanol) (10:1)], which yielded 72 mg of colorless needlesafter repeated crystallization from methanol (overall yield from driedplant material: 0.016% w/w).

[0030] As confirmed by the data summarized in Table 1, betulinic acidhas been reported as noncytotoxic with respect to cultured KB cells.Cytotoxicity of the crude extracts and purified compounds was determinedin a number of cultured human cancer cell lines. Table 1 sets forth thevarious types of cancer cells evaluated. The cells were cultured inappropriate media and under standard conditions. To maintain logarithmicgrowth, the media were changed 24 hours prior to cytotoxic assays. Onthe day of the assay, the cells were harvested by trypsinization,counted, diluted in media, and added to 96-well plates containing testcompounds dissolved in DMSO; the final DMSO concentration was 0.05%.

[0031] The plates were incubated for three days. Following theincubation period, the cells were fixed and stained with sulforhodamineB (SRB) dye. The bound dye was liberated with Tris base, and the OD₅₁₅was measured on an ELISA reader. The growth of the betulinicacid-treated cells was determined by the OD₅₁₅ values, and the growthwas compared to the OD₅₁₅ values of DMSO-treated control cells. Doseresponse studies were performed to generate ED₅₀ values.

[0032] The isolated active compound, betulinic acid (ED₅₀ of 2.0 μg/mlfor MEL-2), has a molecular formula of C₃₀H₄₈O₃, as determined byhigh-resolution mass spectral analysis, a melting point range of292-293° C. (decomposition). The literature melting point range forbetulinic acid is 290-293° C. A mixed melting point range with a knownsample of betulinic acid was not depressed. The optical rotation of thecompound was measured as +7.3° (c=1.2; pyridine) (lit. +7.5°). Theidentity of the isolated compound as betulinic acid was confirmed bycomparing the above physical properties, as well as ¹H-nmr, ¹³C-nmr andmass spectral data of the isolated compound, with physical data andspectra of a known sample of betulinic acid as reported in theliterature.

[0033] To test the in vivo ability of betulinic acid to serve as anantineoplastic agent against malignant melanoma, a series of studies wasperformed with athymic (nude) mice injected subcutaneously with humanmelanoma cells (MEL-2). The initial study investigated the activity ofbetulinic acid against unestablished tumors. Treatment with betulinicacid began on day 1, i.e., 24 hours, following tumor cell injection. Atdoses of 50, 250, and 500 mg/kg (milligram per kilogram) body weight,betulinic acid demonstrated effective inhibition of tumor growth with pvalues of 0.001 for each dose versus a control (FIG. 1). These resultsindicate that betulinic acid can be used to prevent melanoma by topicalapplication of melanoma. Such a discovery is important for individualswho are predisposed to melanoma due to hereditary or environmentalfactors.

[0034] In particular, the data plotted in FIG. 1 was derived fromexperiments wherein four week old athymic mice were injectedsubcutaneously in the right flank with 3.0×10⁸ UISO MEL-2 cells. UISOMEL-2 is a cell line derived from metastatic melanoma from human pleuralfluid. Drug treatment was initiated on the day following tumor cellinjection and continued every fourth day for a total of six doses. Fourcontrol animals received 0.5 ml intraperitoneal (IP) of PVP controlsolution, while treated animals (4 per group) received 50, 250 or 500mg/kg/dose IP betulinic acid/PVP in deionized H₂O. Betulinic acid wascoprecipitated with PVP to increase solubility and bioavailability. Themice were weighed, and the tumors measured with a micrometer every otherday throughout the study. All animals were sacrificed and autopsied onday 33, when the mean tumor volume in the control animals wasapproximately one cm³.

[0035] There was greater inhibition of tumor growth at the highest doseof betulinic acid versus the lowest dose (p=0.04). Toxicity was notassociated with the betulinic acid treatment because toxicity isindicated by loss of body weight or other forms of acute toxicity. Noweight loss was observed.

[0036] Next, in vivo testing of betulinic acid was performed onestablished melanomas. In this study, treatment was withheld until day13, by which time a palpable tumor mass was present in all mice. Asillustrated in FIG. 2, under these conditions betulinic acidsuccessfully abrogated tumor growth (p=0.0001). Furthermore, tumorgrowth did not parallel that of the control (untreated) group even 14days after the termination of treatment.

[0037] In particular, with respect to FIG. 2, four-week-old athymic micewere injected with 5×10⁸ MEL-2 cells subcutaneously in the right flank.Four treatment groups of five mice each were studied. In one group, themice received 250 mg/kg/dose of IP betulinic acid/PVP every third dayfor six total doses initiated the day following tumor cell injection.The control group received 0.5 ml IP saline. A DTIC treatment groupreceived 4 mg/kg/dose IP DTIC every third day from day 13 to day 28 ofthe study. The betulinic acid treatment group received 250 mg/kg/dose IPbetulinic acid/PVP every third day from day 13 to day 27. The controland DTIC-treated mice were sacrificed and autopsied on day 36 due totheir large tumor burden. The remaining mice were sacrificed andautopsied on day 41.

[0038] As illustrated in FIG. 2, the efficacy of betulinic acid also wascompared to DTIC, which is clinically available for the treatment ofmetastatic melanoma. The dose of DTIC, which is limited by toxicity, wasselected to be equivalent to that administered to human patients. Tumorgrowth in the betulinic acid-treated group was significantly less thanthat observed in the DTIC-treated animals (p=0.0001). Compared tocontrols, DTIC produced a significant, but less pronounced, reduction intumor growth, with a p value of 0.01. A fourth group in this study wastreated with a schedule similar to that in the initial study. Underthese conditions, betulinic acid, as demonstrated before, significantlyinhibited tumor development (p=0.0001) and caused a prolonged reductionin tumor growth of up to three weeks following treatment termination.

[0039]FIGS. 4 and 5 illustrate that betulinic acid also showed activityagainst MEL-1 cells. In particular, with respect to FIGS. 4 and 5, fourweek old athymic mice were injected subcutaneously in the right flankwith 5.0×10⁸ UISO MEL-1 cells. Drug treatment was initiated on the dayfollowing tumor cell injection and continued every fourth day for atotal of six doses. Four control animals received 0.5 ml intraperitoneal(IP) saline, while treated animals (4 per group) received 5, 50 or 250mg/kg/dose IP betulinic acid/PVP in dd H₂O. The mice were weighed, andtumors were measured with a micrometer every third day throughout thestudy. Treated animals were sacrificed and autopsied on day 41, when themean tumor volume in the control mice was approximately 0.5 cm³. Thecontrol mice then received six doses of 50 mg/kg every fourth daybeginning day 41 and were sacrificed and autopsied on day 71.

[0040] The results illustrated in FIGS. 4 and 5 with respect to MEL-1cells were similar to the results illustrated in FIGS. 1 and 2.Betulinic acid therefore is active both against MEL-1 and MEL-2 cells.

[0041] The mechanism by which antitumor agents mediated their activityis of great theoretical and clinical importance. Therefore, the mode ofaction by which betulinic acid mediates the melanoma-specific effect wasinvestigated. Visual inspection of melanoma cells treated with betulinicacid revealed numerous surface blebs. This observation, as opposed tocellular membrane collapse, suggested the induction of apoptosis. One ofthe most common molecular and cellular anatomical markers of apoptosisis the formation of “DNA ladders,” which correspond to the products ofrandom endonucleolytic digestion of internucleosomal DNA. Althoughrecent studies have shown that a lack of DNA laddering does notnecessarily indicate a failure to undergo apoptosis, double-strand DNAscission that yields a fragment of about 50 kilobase pairs (Kbp) hasbeen shown to consistently correlate with induction of apoptosis byvarious treatments in a variety of cell lines. Thus, generation of the50 Kbp fragment is a reliable and general indicator of apoptosis.Generation of the fragment occurs upstream of the process leading to DNAladders and represents a key early step in the commitment to apoptosis.

[0042] Therefore, an important feature of the present invention is amethod of analyzing and quantifying the formation of the 50 Kbp fragmentas a biomarker for induction of apoptosis in human cancer cell lines.This method comprises treatment of cells in culture, followed byanalysis of the total cellular DNA content using agarose field-inversiongel electrophoresis. Under these conditions, the 50 Kbp fragment isresolved as a diffuse band. The fraction of the total cellular DNArepresented by the 50 Kbp fragment is determined by densitometry on thecontour of this band.

[0043] To investigate the ability of betulinic acid to induce apoptosis,the above-described method was adapted for use with the MEL-2 cell line.As shown in FIG. 3A, time-dependent formation of a 50 Kbp DNA fragmentwas induced by betulinic acid with MEL-2 cells. Induction was at amaximum after a 56 hour treatment period. After this time period, adecline in the relative amount of the 50 Kbp fragment was observed,probably due to internal degradation. Also observed in the agarose gelwere DNA fragments of about 146 and about 194 Kbp, which are theorizedto be precursors in the process leading to the formation of the 50 Kbpfragment. Additionally, the induction of apoptosis (50 Kbp fragment)mediated by betulinic acid was dose-dependent (FIG. 3B), and the ED₅₀value (about 1.5 μg/ml) observed in the apoptotic response closelyapproximated the ED₅₀ value previously determined for the cytotoxicresponse (Table 1).

[0044] With further respect to FIG. 3A, cultured MEL-2 cells (10⁶ cellsinoculated per 25 cm² flask) were treated with 2 g/ml betulinic acid(200 μg/ml DMSO, diluted 1:100 in media) for 24, 32, 48, 56 and 72hours. After the treatment, the cells were harvested, collected bycentrifugation, then snap frozen in liquid nitrogen for subsequentanalysis. Samples were analyzed on a 1% agarose gel in a Hoefer HE100SuperSub apparatus cooled to 10° C. by a circulating water bath. Theelectrode buffer was 0.5×TBE buffer containing 0.25 μg/ml ethidiumbromide and was circulated during electrophoresis. Each gel included 20μL Sigma Pulse Marker 0.1-200 Kbp DNA size markers. Prior to sampleloading, 50 μL 2% SDS was added to each sample well. Each sample tubewas rapidly thawed, then the pelleted cells were immediately transferredin a volume about 50 μL to the well containing SDS. Each well then wasoverlaid with molten LMP agarose, which was allowed to gel prior toplacing the gel tray in the SuperSub apparatus.

[0045] Electrophoresis was performed at 172 volts for a total of 18hours using two sequential field inversion programs with pulse ramping.The DNA/ethidium bromide fluorescence was excited on a UVtransilluminator and photographed using Polaroid type 55 P/N film. Thenegative was analyzed using a PDI scanning densitometer and Quantity Onesoftware. The intensity of the 50 Kbp fragment was determined bymeasuring the contour optical density (OD×mm²) as a percent of the totaloptical density in the sample lane, including the sample well. Thedecrease in the 50 Kbp band definition caused by internal degradation,and does not represent a reversal of the process.

[0046] With further respect to FIG. 3B, cultured MEL-2 cells weretreated for 56 hours with the following concentrations of betulinicacid: 0, 0.1, 1.0, 2.0, 4.0 and 8.0 μg/ml. The cells were harvested andapoptosis measured as described for FIG. 3A. The experiment was repeatedand a similar dose-response curve was observed (data not shown).

[0047] These data suggest a causal relationship, and it is theorizedthat betulinic acid-mediated apoptosis is responsible for the antitumoreffect observed with athymic mice. Time-course experiments with humanlymphocytes treated in the same manner with betulinic acid atconcentrations of 2 and 20 μg/ml did not demonstrate formation of the 50Kbp fragment (data not shown) indicating the specificity and possiblesafety of the test compound.

[0048] Taking into account a unique in vitro cytotoxicity profile, asignificant in vivo activity, and mode of action, betulinic acid is anexceptionally attractive compound for treating human melanoma. Betulinicacid also is relatively innocuous toxicity-wise, as evidenced byrepeatedly administering 500 mg/kg doses of betulinic acid withoutcausing acute signs of toxicity or a decrease in body weight. Betulinicacid was previously found to be inactive in a Hippocratic screen at 200and 400 mg/kg doses.

[0049] Betulinic acid also does not suffer from the drawback ofscarcity. Betulinic acid is a common triterpene available from manyspecies throughout the plant kingdom. More importantly, a betulinic acidanalog, betulin, is the major constituent of white-barked birch species(up to 22% yield), and betulin can be converted to betulinic acid.

[0050] In addition to betulinic acid, betulinic acid derivatives can beused in a topically applied composition to selectively treat, or preventor inhibit, a melanoma. Betulinic acid derivatives include, but are notlimited to esters of betulinic acid, such as betulinic acid esterifiedwith an alcohol having one to sixteen, and preferably one to six, carbonatoms, or amides of betulinic acid, such as betulinic acid reacted withammonia or a primary or secondary amine having alkyl groups containingone to ten, and preferably one to six, carbon atoms.

[0051] Another betulinic acid derivative is a salt of betulinic acid.Exemplary, but nonlimiting, betulinic acid salts include an alkali metalsalt, like a sodium or potassium salt; an alkaline earth metal salt,like a calcium or magnesium salt; an ammonium or alkylammonium salt,wherein the alkylammonium cation has one to three alkyl groups and eachalkyl group independently has one to four carbon atoms; or transitionmetal salt.

[0052] Other betulinic acid derivatives also can be used in thecomposition and method of the present invention. One other derivative isthe aldehyde corresponding to betulinic acid or betulin. Anotherderivative is acetylated betulinic acid, wherein an acetyl group ispositioned at the hydroxyl group of betulinic acid.

[0053] In particular, betulinic acid derivatives have been synthesizedand evaluated biologically to illustrate that betulinic acid derivativespossess selective antitumor activity against human melanoma cells linesin vitro. It has been demonstrated that modifying the parent structureof betulinic acid provides numerous betulinic acid derivatives that canbe deused to prevent or inhibit malignant tumor growth, especially withrespect to human melanoma. The antitumor activity of betulinic acidderivatives is important because betulinic acid, although exhibiting ahighly selective activity against melanomas, also possesses a low watersolubility. The low water solubility of betulinic acid, however, can beovercome by providing an appropriate derivative of betulinic acid.Modifying the parent structure betulinic acid structure also can furtherimprove antitumor activity against human melanoma.

[0054] An examination of the structure of betulinic acid, i.e., compound(1), reveals that betulinic acid contains three positions, i.e., theC-3, C-20, and C-28 positions, where functional groups can beintroduced. In addition, the introduced functional groups, if desired,then can be modified. Through a series of reactions at these threepositions, a large number of betulinic acid derivatives were preparedand evaluated for bioefficacy against a series of human tumor celllines, especially against human melanoma cell lines.

[0055] With respect to modifications at the C-3 position of betulinicacid, the hydroxyl group at the C-3 position can be converted to acarbonyl group by an oxidation reaction. The resulting compound isbetulonic acid, i.e., compound (2). The ketone functionality ofbetulonic acid can be converted to oxime (3) by standard syntheticprocedures. Furthermore, a large number of derivatives (4) can beprepared through substitution reactions performed on the hydroxyl groupof oxime (3), with electrophiles, as set forth in equation (a):

[0056] wherein R_(a)═H or C₁-C₁₆ alkyl, or R_(a)═COC₆H₄X, wherein X═H,F, Cl, Br, I, NO₂, CH₃, or OCH₃, or R_(a)═COCH₂Y, wherein Y═H, F, Cl,Br, or I, or R_(a)═CH₂CHCH₂ or CH₂CCR₁, wherein R₁ is H or C₁-C₆ alkyl.When R_(a) is C₁-C₁₆ alkyl, preferred alkyl groups are C₁-C₆ alkylgroups.

[0057] The ketone functionality of betulonic acid can undergo areductive amination reaction with various aliphatic and aromatic aminesin the presence of sodium cyanoborohydride (NaBH₃CN) to provide thecorresponding substituted amines (5) at the C-3 position, as set forthin equation (b).

[0058] wherein R_(b)═H or C₁-C₁₀ alkyl, or R_(b)═C₆H₄X. A primary aminederivative, i.e., R_(b)═H, at the C-3 position can be reacted with aseries of acyl chlorides or anhydrides, or alkyl halides, to provideamides and secondary amines (6), respectively, as set forth in equation(c).

[0059] wherein R_(c)═COC₆H₄X, or R_(c)═COCH₂Y, or R_(c)═CH₂CHCH₂ orCH₂CCR₁.

[0060] The ketone functionality of betulonic acid can react with aseries of lithium acetylides (i.e., LiC≡CR₁) to provide alkynyl alcoholderivatives (7) at the C-3 position. Based on the chemical reactivityand the stereoselectivity of the betulonic acid structure, α-alkynylsubstituted β-hydroxyl alkynyl betulinic acid are the major products ofthe reaction, as set forth in equation (d).

[0061] wherein R_(d)═CCR₁, wherein R₁ is H or C₁-C₆ alkyl.

[0062] A number of esters also can be prepared by reacting the hydroxylgroup of betulinic acid with a variety of acyl chlorides or anhydrides(8), as set forth in equation (e).

[0063] wherein R_(e)═R₁CO or XC₆H₄CO.

[0064] With respect to modification at the C-28 position, the carboxylgroup of betulinic acid can be converted to a number of esters (9) andamides (10) by reaction with an alcohol or an amine, respectively, asset forth in equations (f) and (g). Depending on the types of functionalgroups present on the alcohols or amines, additional structuralmodification are possible. The carboxyl group also can be converted to asalt, in particular an alkali metal salt, an alkaline earth salt, anammonium salt, an alkylammonium salt, a hydroxyalkyl ammonium salt, or atransition metal salt.

[0065] wherein R_(f)═C₁-C₁₀ alkyl, phenyl, substituted phenyl (C₆H₄X),or CH₂CCR₁.

[0066] The activated C-28 hydroxyl group of betulin can undergosubstitution reactions, like SN-2 type reactions, with nucleophiles toprovide an amino (11) or an ether derivative (12), as set forth inequations (h) and (i).

[0067] wherein R_(g)═H or C₁-C₁₆ alkyl, or R_(g)═C₆H₄X, and whereinR_(h)═C₁-C₁₆ alkyl or C₆H₄X.

[0068] The hydroxyl group at the C-28 position can be oxidized to yieldan aldehyde, which in turn can react with hydroxylamine to provide ahydroxyloxime compound. The hydroxyloxime can react with a variety ofelectrophiles to provide the oxime derivatives (13), as set forth inequation (j).

[0069] wherein R_(i)═H or C₁-C₁₆ alkyl, or R_(i)═COC₆H₄X, orR_(i)═COCH₂Y, or R_(i)═CH₂CHCH₂ or CH₂CCR₁.

[0070] The aldehyde at the C-28 position also can react with a series oflithium acetylide compounds to yield a variety of alkynyl betulinderivative (14), as set forth in equation (k).

[0071] wherein R_(j)═CCR₁, wherein R₁═H or C₁-C₆ alkyl.

[0072] With respect to modifications at the C-20 position, the isoprenylgroup at the C-20 position can be ozonized to yield a ketone (15) atC-20 position, as set forth in equation (1). A variety of reactionsperformed on the ketone functionality can provide a series of differentderivatives. For example, the ketone functionality of compound (15) canbe easily converted to a variety of oximes. Furthermore, a number ofadditional oxime derivatives (16) can be prepared through substitutionreactions at the hydroxyl group of the hydroxyloxime with electrophiles,as set forth in equation (m).

[0073] wherein R_(k)═H or C₁-C₁₆ alkyl, or R_(k)═COC₆H₄X orR_(k)═COCH₂Y, or R_(k)═CH₂CHCH₂ or CH₂CCR₁.

[0074] The ketone functionality also can undergo a reductive aminationreaction with a series of aliphatic and aromatic amines in the presenceof NaBH₃CN to provide a corresponding substituted amine (17) at the C-20position, as set forth in equation (n).

[0075] wherein R₁═C₁-C₁₆ alkyl, or R₁═C₆H₄X, or R₁═COC₆H₄X, orR₁═COCH₂Y, or R₁═CH₂CHCH₂ or CH₂CCR₁.

[0076] The ketone can be reacted with a series of lithium acetylides toprovide alkynyl alcohol derivatives (18) at the C-20 position, as setforth in equation (o).

[0077] wherein, R_(m)═CCR₁.

[0078] The ketone further can be reduced to a secondary alcohol (19) toreact with an acyl chloride to provide a series of esters (20) at theC-20 position, as set forth in equation (p).

[0079] wherein R_(n)═H, C₁-C₁₆ alkyl, CH₂CCR₁, or R_(n)═CH₃CO orXC₆H₄CO.

[0080] In addition, a number of different derivatives can be preparedthrough a combinatorial chemical approach. For example, as set forthbelow, in the preparation of oximes at the C-20 position, a number ofelectrophiles, e.g., a variety of alkyl halides, can be added togetherin one reaction vessel containing the hydroxyloxime to provide a mixtureof betulinic acid derivatives. Each reaction product in the mixture canbe isolated by using semi-preparative HPLC processes using appropriateseparation conditions, then submitted for bioassay.

[0081] wherein P is a protecting group for the secondary alcoholfunctionality.

[0082] A low temperature reaction of betulonic acid with a mixture oflithium acetylides in a single reaction vessel, as set forth below,yielded a mixture of alkynyl alcohols at the C-3 position. Eachcomponent in the mixture can be isolated by using semi-preparative HPLCprocesses using appropriate separation conditions, then submitted forbioassay.

[0083] In order to demonstrate that betulinic acid derivatives have apotent bioefficacy, various derivatives were subjected to a series ofbiological evaluation tests. The biological evaluation of thederivatives focused on the activity against human melanoma cell lines.In particular, the following betulinic acid derivatives were preparedand tested for cytotoxicity profile against human melanoma cell linesand against a number of selected nonmelanoma cell lines. The results aresummarized in Table 2. The data shows that some hydrogenatedderivatives, i.e., compounds 5 and 11, are less active thannonhydrogenated derivatives 13 and 10, respectively. However, otherhydrogenated derivatives, i.e., compounds 7 and 6, showed a comparablebiological activity to nonhydrogenated derivatives 2 and 8,respectively. Therefore, it is possible to optimize the modification atthe C-20 position to yield more potent betulinic acid derivatives. Table3 contains a summary of data showing the effect of hydrogenation at theC-20 position. TABLE 2 Cytotoxicity Data of Betulinic Acid Derivatives

ED₅₀ [μg/mL] (Std. Dev.) MALE- Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 3MLOX KB  1 O═ CHO CH₂═C(CH₃)₂ 7.4 (2.4) >20 3.2 (1.2) >20 18.5 12.9  2HO—N═ COOH CH₂═C(CH₃)₂ 2.4 (0.3) 14.8 (2.0) 1.9 (1.0) 15.8 9.1 >20  3CH₃O—N═ CHNOCH₃ CH₂═C(CH₃)₂ >20 >20 >20 >20 >20 20  4 HO—N═ CHNOHCH₂═C(CH₃)₂ 2.2 (0.7) 11.9 (2.7) 1.4 (0.6) 17.5 4.1 3.3  5 CH₃O—N═ COOHC(CH₃)₃ >20 >20 >20 >20  6 O═ COOH C(CH₃)₃ 0.7 (0.6) 10.8 (2.6) 0.9(0.4) 20 (Dihydrobetulonic acid)  7 HO—N═ COOH C(CH₃)₃ 2.2 (0.3) 13.1(1.1) 1.6 (1.1) 13.9  8 O═ COOH CH₂═C(CH₃)₂ 0.9 (0.8) 15.3 (3.4) 0.4(0.1) 20 6.9 2.5 (Betulonic acid)  9 H₂N— COOH CH₂═C(CH₃)₂ 1.3 (0.4) 5.2(2.6) 1.3 (0.5) 3.1 10 HO— COOH CH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5) 1.0(0.3) 17.6 (0.5) >20 >20 (Betulinic acid) 11 HO— COOH C(CH₃)₃5.8 >20 >20 (Dihydrobetulinic acid) 12 HO— CH₂OH CH₂═C(CH₃)₂ >20 >20 >20(Betulin) 13 CH₃O—N═ COOH CH₂═C(CH₃)₂ 8.3 >20 4.3 14 HO— COOCH₃CH₂═C(CH₃)₂ 8.3 12.5 11.8 (Methyl betulinate) 15 HO— CH₃ CH₂═C(CH₃)₂17.6 15.6 >20 (Lupeol) 16 C₆H₄COO— CH₃ CH₂═C(CH₃)₂ >20 >20 >20 (Lupeolbenzoate)

[0084] TABLE 3 Cytotoxicity Data of Betulinic Acid Derivatives (Effectof Hydrogenation at C-20)

ED₅₀ [μg/mL] (Std. Dev.) MALE- Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 3MLOX KB 13  CH₃O—N═ COOH CH₂═C(CH₃)₂ 8.3 >20 4.3 5 CH₃O—N═ COOHC(CH₃)₃ >20 >20 >20 >20 10  HO— COOH CH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5)1.0 (0.3) 17.6 (0.5) >20 >20 (Betulinic acid) 11  HO— COOH C(CH₃)₃5.8 >20 >20 (Dihydrobetulinic acid) 2 HO—N═ COOH CH₂═C(CH₃)₂ 2.4 (0.3)14.8 (2.0) 1.9 (1.0) 15.8 9.1 >20 7 HO—N═ COOH C(CH₃)₃ 2.2 (0.3) 13.1(1.1) 1.6 (1.1) 13.9 8 O═ COOH CH₂═C(CH₃)₂ 0.9 (0.8) 15.3 (3.4) 0.4(0.1) 20 6.9 2.5 (Betulonic acid) 6 O═ COOH C(CH₃)₃ 0.7 (0.6) 10.8 (2.6)0.9 (0.4) 20 (Dihydrobetulonic acid)

[0085] The modification of betulinic acid at the C-3 position showedthat all compounds, except methoxy oxime 13, expressed a comparablebiological activity toward melanoma cell lines (Table 4). Amino compound9 exhibited an improved cytotoxicity compared to betulinic acid 10.Compounds. 2, 8, and 13 showed a decrease in selective cytotoxicitycompared to betulinic acid. TABLE 4 Cytotoxicity Data of Betulinic AcidDerivatives (Modification at C-3 Position)

ED₅₀ [μg/mL] (Std. Dev.) Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOXKB 10  HO— COOH CH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5) 1.0 (0.3) 17.6(0.5) >20 >20 (Betulinic acid) 8 O═ COOH CH₂═C(CH₃)₂ 0.9 (0.8) 15.3(3.4) 0.4 (0.1) 20 6.9 2.5 (Betulonic acid) 2 HO—N═ COOH CH₂═C(CH₃)₂ 2.4(0.3) 14.8 (2.0) 1.9 (1.0) 15.8 9.1 >20 13  CH₃O—N═ COOH CH₂═C(CH₃)₂8.3 >20 4.3 9 H₂N— COOH CH₂═C(CH₃)₂ 1.3 (0.4)  5.2 (2.6) 1.3 (0.5)  3.1

[0086] With respect to modifications at the C-28 position, the freecarboxylic acid group at C-28 position is important with respect toexpression of biological activity (Table 5). However, it is unknownwhether the size or the strength of hydrogen bonding or thenucleophilicity of the C-28 substituents is responsible for thebiological effect. TABLE 5 Cytotoxicity Data of Betulinic AcidDerivatives (Modification at C-28 Position) ED₅₀ [μg/mL] (Std. Dev.)Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOX KB 12 HO— CH₂OHCH₂═C(CH₃)₂ >20 >20 >20 (Betulin) 10 HO— COOH CH₂═C(CH₃)₂    1.2 (0.1)13.2 (1.5) 1.0 (0.3) 17.6 (0.5) >20 >20 (Betulinic acid) 14 HO— COOCH₃CH₂═C(CH₃)₂    8.3 12.5 11.8 (Methyl betulinate) 15 HO— CH₃ CH₂═C(CH₃)₂  17.6 15.6 >20 (Lupeol)

[0087] The biological activity changes attributed to oximes isillustrated in Table 6. The hydroxyloxime 4 improved the cytotoxicityprofile, although selectivity was lost. It appears that the size of thesubstituent and its ability to hydrogen bond may influence theexpression of the biological activity. TABLE 6 Cytotoxicity Data ofBetulinic Acid Derivatives (Effect by Oximes) ED₅₀ [μg/mL] (Std. Dev.)Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOX KB 12  HO— CH₂OHCH₂═C(CH₃)₂ >20 >20 >20 (Betulin) 1 O═ CHO CH₂═C(CH₃)₂ 7.4 (2.4) >20 3.2(1.2) >20 18.5 12.9 4 HO—N═ CHNOH CH₂═C(CH₃)₂ 2.2 (0.7) 11.9 (2.7) 1.4(0.6) 17.5 4.1 3.3 3 CH₃O—N═ CHNOCH₃ CH₂═C(CH₃)₂ >20 >20 >20 >20 >20 20

[0088] The above tests show that modifying the parent structure ofbetulinic acid can provide derivatives which can be used as potentantitumor drugs against melanoma. Betulinic acid derivatives having acomparable or better antitumor activity than betulinic acid againsthuman melanoma have been prepared. In addition, even though betulinicacid has a remarkably selective antitumor activity, betulinic acid alsohas a poor solubility in water. The low solubility of betulinic acid inwater can be overcome by introducing an appropriate substituent on theparent structure, which in turn can further improve selective antitumoractivity. In addition, because the parent compound, betulinic acid, hasshown to possess anti-HIV activity, the derivatives also can bedeveloped as potential anti-HIV drug candidates.

What is claimed is:
 1. A composition for treating melanoma comprisingbetulinic acid modified at the C-3 position.
 2. The composition of claim1 wherein the modified betulinic acid has the structure:

wherein R_(a) is H, C₁-C₁₆ alkyl, COC₆H₄X, COCH₂Y, CH₂CHCH₂, or CH₂CCR₁,and wherein X is H, F, Cl, Br, I, NO₂, CH₃, or OCH₃, Y is H, F, Cl, Br,or I, and R₁ is H or C₁-C₆ alkyl.
 3. The composition of claim 1 whereinthe modified betulinic acid has the structure:

wherein R_(b) is H, C₁-C₁₀ alkyl, C₆H₄Y, COC₆H₄X, COCH₂Y, CH₂CHCH, orCH₂CCR₁, and wherein X is H, F, Cl, Br, I, NO₂, CH₃, or OCH₃, Y is H, F,Cl, Br, or I, and R₁ is H or C₁-C₆ alkyl.
 4. The composition of claim 1wherein the modified betulinic acid has the structure:

wherein R_(d) is CCR₁, and wherein R₁ is H or C₁-C₆ alkyl.
 5. Thecomposition of claim 1 wherein the modified betulinic acid has thestructure:

wherein R_(e) is R₁CO or COC₆H₄X, wherein R₁ is H or C₁-C₆ alkyl, and Xis H, F, Cl, Br, I, NO₂, CH₃, or OCH₃.
 6. A composition for treatingmelanoma comprising betulinic acid modified at the C-28 position.
 7. Thecomposition of claim 6 wherein the modified betulinic acid has thestructure:

wherein R_(f) is C₁-C₁₀ alkyl, phenyl, C₆H₄X, or CH₂CCR₁, wherein X isH, F, Cl, Br, I, NO₂, CH₃, or OCH₃, and R₁ is H or C₁-C₆ alkyl.
 8. Thecomposition of claim 6 wherein the modified betulinic acid has thestructure:

wherein R_(g) is H, C₁-C₆ alkyl, or C₆H₄X, R_(h) is C₁-C₁₆ alkyl orC₆H₄X, and wherein X is H, F, Cl, Br, I, NO₂, CH₃, or OCH₃.
 9. Thecomposition of claim 6 wherein the modified betulinic acid has thestructure:

wherein R_(i) is H, C₁-C₁₆ alkyl, COC₆H₄X, COCH₂Y, CH₂CHCH₂, or CH₂CCR₁,and wherein X is H, F, Cl, Br, I, NO₂, CH₃, or OCH₃, and R₁ is H orC₁-C₆ alkyl.
 10. The composition of claim 6 wherein the modifiedbetulinic acid has the structure:

wherein R_(j) is CCR₁, and wherein R₁ is H or C₁-C₆ alkyl.
 11. Acomposition for treating melanoma comprising betulinic acid modified atthe C-20 position.
 12. The composition of claim 11 wherein the modifiedbetulinic acid has the structure:


13. The composition of claim 11 wherein the modified betulinic acid hasthe structure:

wherein R_(k) is H, C₁-C₁₆ alkyl, COC₆H₄X, COCH₂Y, CH₂CHCH₂, CH₂CCR₁,and wherein X is H, F, Cl, Br, I, NO₂, CH₃, or OCH₃, Y is H, F, Cl, Br,or I, and R₁ is H or C₁-C₆ alkyl.
 14. The composition of claim 11wherein the modified betulinic acid has the structure:

wherein R₁ is C₁-C₁₆ alkyl, C₆H₄X, COC₆H₄X, COCH₂Y, CH₂CHCH₂, orCH₂CCR₁, and wherein X is H, F, Cl, Br, I, NO₂, CH₃, or OCH₃, Y is H, F,Cl, Br, or I, and R₁ is H or C₁-C₁₆ alkyl.
 15. The composition of claim11 wherein the modified betulinic acid has the structure:

wherein R_(m) is CCR₁, and R₁ is H or C₁-C₆ alkyl.
 16. The compositionof claim 11 wherein the modified betulinic acid has the structure:

wherein R_(n) is H, C₁-C₁₆ alkyl, CH₂CCR₁, CH₃CO, or COC₆H₄X, andwherein R₁ is H or C₁-C₆ alkyl, and X is H, F, Cl, Br, I, NO₂, CH₃, orOCH₃.
 17. A composition for treating melanoma comprising a compoundhaving a structure:

wherein R₁ is selected from the group consisting of O, HO—N, CH₃O—N,H₂N, HO, and C₆H₄CO₂, R₂ is selected from the group consisting of CHO,CO₂H, CHNOCH₃, CHNOH, and CH₂OH, and R₃ is C(CH₃)₃ or CH₂═C(CH₃)₂.
 18. Amethod of inhibiting growth of a melanoma comprising topically applyinga therapeutically effective amount of the composition of claim 1 to themelanoma.
 19. A method of inhibiting growth of a melanoma comprisingtopically applying a therapeutically effective amount of the compositionof claim 6 to the melanoma.
 20. A method of inhibiting growth of amelanoma comprising topically applying a therapeutically effectiveamount of the composition of claim 11 to the melanoma.
 21. A method ofinhibiting growth of a melanoma comprising topically applying atherapeutically effective amount of the composition of claim 17 to themelanoma.
 22. A method of preventing melanoma comprising topicallyapplying a composition of claim 1 to skin.
 23. A method of preventingmelanoma comprising topically applying a composition of claim 6 to skin.24. A method of preventing melanoma comprising topically applying acomposition of claim 11 to skin.
 25. A method of preventing melanomacomprising topically applying a composition of claim 17 to skin.
 26. Amethod of preventing melanoma comprising topically applying betulinicacid to skin.